Elsevier

Oral Oncology

Volume 44, Issue 12, December 2008, Pages 1139-1146
Oral Oncology

Aberrant expression of β-dystroglycan may be due to processing by matrix metalloproteinases-2 and -9 in oral squamous cell carcinoma

https://doi.org/10.1016/j.oraloncology.2008.02.016Get rights and content

Summary

Dystroglycan (DG), a non-integrin adhesion molecule, is formed by two subunits, α- and β-DG, which bind to extracellular matrix molecules and cytoskeleton. DG expression is frequently reduced in human cancers and has been related to tumor grade and aggressiveness. The exact proteolytic processing of β-DG remains largely unknown. In this study, we investigated the correlation of β-DG degradation with invasiveness in oral squamous cell carcinoma (OSCC) and its possible processing by matrix metalloproteinases (MMP). Immunohistochemical staining was used to assess β-DG expression in 60 cases of OSCC. The effects of the MMP inhibitor 1,10-phenanthroline on tumour cell invasion and β-DG degradation were investigated using in vitro invasion assays and immunoblot analysis. Co-immunoprecipitation and N-terminal sequencing were performed to determine the possible cleavage site of β-DG by MMP. The α- and β-DG expression was reduced or lost in OSCC. In four cell lines studied (SCC-4, SCC-9, SCC-15 and SCC-25), Western blot revealed a 30 kDa fragment of β-dystroglycan (β-DG30) in addition to β-DG itself. β-DG degradation was almost abolished using 1,10-phenanthroline and there was a significant decrease in tumor cell invasion. The N-terminal sequence of β-DG30 was detected as Ile-Asn-Thr-Asn, or Ile-Val-Thr-Gln. We conclude that β-DG degradation may play a role both in OSCC invasion and metastasis. MMP activity seems to be one mechanism for β-DG processing into β-DG30.

Introduction

It is well known that cell–cell and cell–extracellular matrix (ECM) interactions have a pivotal function in the development and maintenance of normal cell architecture. Abnormalities of the interactions between tumour cells and ECM proteins are thought to be common features of malignant tumors and crucial step for initiation of tumor metastasis.1 During the cell–cell and/or cell–ECM interactions, cell adhesion molecules are thought to play many important roles, including attachment of epithelial cell to the basal lamina, information exchange, cytoskeleton organization, and signal transduction. However, studies on cell–ECM interactions have largely focused on the integrins, an extensively characterized family of heterodimeric receptors.1, 2 In recent years, the role of non-integrin receptors has gained much attention in cancer biology.

Dystroglycan (DG) is a non-integrin adhesion molecule expressed by a variety of tissues, interacting with extracellular proteins including laminin, perlecan and agrin, and membrane proteins such as the neurexins. DG is a product of a single gene (DAG1), but the primary peptide is post-translationally cleaved into two protein subunits (α and β), which interact to form a functional non-covalent complex.3 DG was firstly isolated from skeletal muscle, and has been mainly studied for its role in skeletal muscle cell stability and its alterations in muscular diseases, such as dystrophies. In recent years, however, DG has been reported to play important roles in regulating basal membrane morphogenesis, cytoskeletal organization, cell polarization, cell growth, signal transduction, maintenance of tissue integrity in epithelial cells.4, 5, 6 These findings might implicate DG in cancer biology. In fact, abnormalities in DG expression in human breast, colon, prostate, and oral cancers have been described.7, 8, 9, 10 These studies confirmed that DG expression was reduced or lost in primary and metastatic cancers with no rearrangement of DG mRNA. However, the role of β-DG degradation and its mechanisms were poorly understood.

It is well known that matrix metalloproteinases (MMPs) are an important group of proteolytic enzymes that are capable of degrading the basement membrane, as well as certain cell membrane proteins. MMPs are produced by cancer cells or through the induction of surrounding stromal cells, and play crucial roles in the degradation and remodeling of ECM and tumour angiogenesis.11 Recently, Yamada et al. found that the processing of β-DG by MMP could cause the disruption of the link between ECM and cell membrane via the dystroglycan complex in muscle cells.12 This provides a possible mechanism underlying the degradation of β-DG in cancer.

Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy worldwide and often invades tissues locally and metastasises to cervical lymph nodes. The purpose of this study was to investigate the expression of α-, β-DG in OSCC, the implications of aberrant β-DG in the migration and invasion of cancer cells, and the possible role of MMPs in the proteolytic processing of β-DG in OSCC.

Section snippets

Antibodies and reagents

Mouse monoclonal anti-human beta-DG antibody (clone 43DAG/8D5) was purchased from Novocastra Laboratories Ltd., Newcastle, UK. Two mouse anti-α-DG monoclonal antibodies were used: VIA4-1 (Upstate Lab, USA) and anti-α-DG (IgM, US Biological, USA). Mouse monoclonal anti-human MMP-2 (8B4) and MMP-9 (2C3) antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. 1,10-Orthophenanthroline monohydrate (Sigma–Aldrich, Poole, UK) was used as matrix metalloprotease (MMP)

Abnormal expression of α, β-DG in oral cancer

In normal oral epithelium, both α- and β-DG were found to localize in the basal cell layers of the squamous epithelium, in muscle sarcolemma and connective tissue blood vessels (Fig. 1A and B). The DG expression was completely altered in OSCC. A nearly total lack of α-DG staining was found in all OSCC samples (Fig. 1C and D). Unlike the specific expression pattern in normal epithelium, however, the β-DG staining in cancer tissue was highly variable. The β-DG expression was frequently reduced or

Discussion

Dystroglycan is a cellular receptor expressed by a variety of tissues, interacting with extracellular proteins including laminin, perlecan and agrin, and membrane proteins such as the neurexins.3, 13 Specific changes in DG expression have been described in human breast, colon, prostate and head and neck cancers.7, 8, 9, 10 However to our knowledge, there are no studies that have investigated the aberrant expression of DG and the possible mechanisms underlying the degradation of β-DG in oral

Conflict of Interest Statement

None declared.

Acknowledgements

This work was supported by the Grant from Royal College of Surgeons of England awarded to Professor P.A. Brennan, and the Grant from National Natural Science Foundation of China (No. 30772435) awarded to Dr. Zheng-Jun Shang. The authors also thank Dr. Chu Fu Lien for his kind help in the experiment.

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